Dr. Bluestone's laboratory has developed a novel class of anti-CD3-based immunosuppressant that was effective at suppressing immune response without the severe side effects associated with the use of conventional anti-CD3 mAb therapy. The novel anti-CD3 mAbs appear to induce immune tolerance by anergizing the pathogenic Th1 cells, and promote the opposing Th2 response. Biochemical analysis showed identical early TCR signaling patterns in both subsets of T cells similar to that observed in T cells treated with altered peptide ligands. It is hypothesized that imbalanced signal is responsible for the differential activity and may be a common mechanism to regulate T cell activation and differentiation in vivo. The goal of this proposed study is to is to define the signaling mechanisms that regulate these processes. The first aim of this study is to further map signaling abnormality in T cells treated with the novel anti-CD3 mAb using conventional biochemical approaches. The molecules to be analyzed include Ick, fyn, TCR zetu, JNK, and p38 kinase. The second aim is focused on defining the minimal signaling requirements for anergy induction and Th differentiation using genetic approaches. Altered forms of the above signaling molecules will be introduced into T cells in vitro to either block or enhance individual signaling pathways and effects of such manipulation on anergy induction or Th differentiation will be analyzed. A newly developed retroviral gene transduction system will be used to introduce these genes into either naive or cloned T cells. The results from these experiments will provide information on the functional outcome of differential TCR signaling, which cannot be obtained using transformed T cell lines. As this novel class of anti-CD-3 mAb moves into clinical trials, there is an urgent need to understand the molecular basis of its in vivo effect. The information will be invaluable in designing new safer and more specific therapeutic avenues for preventing transplant rejection and treating autoimmune disorders.